citric acid biotechnology

Citric Acid Biotechnology

Citric AcidBiotechnology

BJRN KRISTIANSEN

Borregaard Industries Ltd, Norway

MICHAEL MATTEY

Department of Bioscience and Biotechnology, University of Strathclyde, UK

JOAN LINDEN

Gluppevelen 15, 1614 Fredikstad, Norway

UK Taylor & Francis Ltd, 1 Gunpowder Square, London EC4A 3DFUSA Taylor & Francis Inc., 325 Chestnut Street, 8th Floor, Philadelphia, PA 19106

This edition published in the Taylor & Francis e-Library, 2002.

Copyright Taylor & Francis 1999All rights reserved. No part of this publication may be reproduced, stored in a retrievalsystem, or transmitted in any form or by any means, electronic, electrostatic, magnetictape, mechanical, photocopying, recording or otherwise, without the prior permission ofthe copyright owner.

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vContents

1 A Brief Introduction to Citric Acid Biotechnology page 11.1 Citric acid from lemons 11.2 Synthetic citric acid 21.3 Microbial citric acid 21.4 Citric acid by the surface method 31.5 The submerged process for production of citric acid 41.6 Continuous and immobilized processes 51.7 Yeast based processes 61.8 The koji process 71.9 Uses of citric acid 71.10 Effluent disposal 81.11 Conclusions 81.12 References 9

2 Biochemistry of Citric Acid Accumulation by Aspergillus niger 112.1 Introduction 112.2 Glucose catabolism in A. niger and its regulation 122.3 Regulation of citric acid biosynthesis 192.4 Role of citrate breakdown in citrate accumulation 212.5 Export of citric acid from A. niger 242.6 References 25

3 Biochemistry of Citric Acid Production by Yeasts 333.1 Introduction 333.2 Synthesis of citric acid from n-alkanes 353.3 Synthesis of citric acid from glucose 463.4 Conclusions 503.5 References 50

4 Strain Improvement 554.1 Introduction 554.2 General aspects of strain improvement 55

Contentsvi

4.3 Isolation of recombinant strains using the parasexual cycle in A. niger 604.4 Genetic engineering 614.5 Concluding remarks 644.6 Acknowledgements 654.7 References 65

5 Fungal Morphology 695.1 Introduction 695.2 Factors affecting Aspergillus niger morphology in submerged culture 695.3 Effect of agitation 705.4 Effect of nutritional factors 745.5 Effect of inoculum 825.6 Conclusions and perspectives 825.7 References 82

6 Redox Potential in Submerged Citric Acid Fermentation 85Nomenclature 85

6.1 Introduction 856.2 Overview 866.3 Theory 876.4 Measurement of redox potential 886.5 Significance of redox potential 896.6 Redox potential in citric acid fermentation 916.7 Regulation of the redox potential 956.8 Regulation of redox potential in citric acid fermentation 956.9 Scale-up based on redox potential 1016.10 Conclusions 1026.11 References 103

7 Modelling the Fermentation Process 1057.1 Introduction 1057.2 Aspergillus based models 1077.3 Yeast based models 1137.4 Conclusion 1197.5 References 119

8 Mass and Energy Balance 121Nomenclature 121

8.1 Introduction 1228.2 Metabolic description of A. niger growth 1238.3 Mass and energy balances 1258.4 Kinetics of growth and citric acid production by A. niger 1288.5 Carbon and available electron balances 1308.6 Conclusion 1318.7 References 132

9 Downstream Processing in Citric Acid Production 1359.1 Pretreatment of fermentation broth 1359.2 Precipitation 136

Contents vii

9.3 Solvent extraction 1399.4 Adsorption, absorption and ion exchange 1429.5 Liquid membranes 1439.6 Electrodialysis 1449.7 Ultrafiltration 1459.8 Immobilization of micro-organisms 1469.9 References 146

10 Fermentation Substrates 14910.1 Introduction 14910.2 Molasses 15010.3 Refined or raw sucrose 15610.4 Syrups 15610.5 Starch 15710.6 Hydrol 15710.7 Alkanes 15710.8 Oils and fats 15810.9 Cellulose 15810.10 Other medium redients 15810.11 Conclusion 15910.12 References 159

11 Design of an Industrial Plant 161Nomenclature 161

11.1 Design of an industrial plant 16311.2 Data required 16311.3 Design basis 16511.4 Scope definition 16711.5 Process package 16711.6 Raw material 16911.7 Substrate preparation 16911.8 Fermentation 17011.9 Design of a stirred tank reactor 17111.10 Airlift and bubble column reactors 17411.11 Product isolation 17611.12 Cell removal 17711.13 Purification 17811.14 Crystallization stages 18211.15 Product packaging 18311.16 Effluent and by-products 18311.17 In conclusion 18311.18 References 184

Index 187

Contributors

Ho Ai Meng AmyBlk 135 Pasir Ris Street 11, # 06-239, Singapore 510135

Marin BerovicDepartment of Chemistry and Biochemical Engineering, National Chemistry Laboratoryfor Biotechnology and Industrial Mycology, 1115 Slo, Ljubljana, Hajdrihova 19 POB 30,Slovenia

Pawel GluszcaDepartment of Bioprocess Engineering, Technical University of Lodz, Wolczanska 17590-924 Lodz, Poland

Bjrn KristiansenBorregaard Industries Ltd, PO Box 162, 1701 Sarpsborg, Norway

Liliana KrzystekDepartment of Bioprocess Engineering, Technical University of Lodz, Wolczanska 17590-924 Lodz, Poland

Christian KubicekInstitute for Biochemical Technology and Microbiology, University of TechnologyGetreidemarkt 9/1725, A-1060 Wien, Austria

Staniskaw LedakowiczDepartment of Bioprocess Engineering, Technical University of Lodzul, Wolczanska 17590-924 Lodz, Poland

Wladyslaw LesniakFood Biotechnology Department, Academy of Economics, Komandorska 118/120 PL 53-345 Wroclaw, Poland

Michael MatteyDepartment of Bioscience and Biotechnology, University of Strathclyde, Todd Centre 33Taylor Street, Glasgow G4 0NR

Maria Papagianni8 Kamvounion Street, 54 621 Thessaloniki, Greece

George RuijterSection of Molecular Genetics of Industrial Microorganisms, Wageningen AgriculturalUniversity, Dreijentlaan 2, 6703 HA Wageningen, The Netherlands

Jacobus van der MerweNCP, Project Engineering Division, PO Box 494, Germiston 1400, South Africa

Jaap VisserSection of Molecular Genetics of Industrial Microorganisms, Wageningen AgriculturalUniversity, Dreijentlaan 2, 6703 HA Wageningen, The Netherlands

Frank WaymanDepartment of Bioscience and Biotechnology, University of Strathclyde, Todd Centre 33Taylor Street, Glasgow G4 0NR

Markus WolschekInstitute for Biochemical Technology and Microbiology, University of TechnologyGetreidemarkt 9/1725, A-1060 Wien, Austria

Contributorsx

11

A Brief Introduction to Citric AcidBiotechnology

MICHAEL MATTEY AND BJRN KRISTIANSEN

1.1 Citric acid from lemons

They are going to be squeezed, as a lemon is squeezeduntil the pips squeak. My only doubt isnot whether we can squeeze hard enough, but whether there is enough juice.

(Sir Eric Geddes, 1918)It is probably no more than a coincidence that Sir Eric Geddes uttered his now famousphrase at the time that the industrial production of citric acid by fungal fermentation wasbeing developed to circumvent the high price and lack of availability of lemon juice. However,the association of taxation and squeezing lemons is appropriate, as the history of citric acidreflects the politics and economics of the era as well as the science. Indeed the productionof citric acid is a classical biotechnology phenomenon, where the science, though important,is secondary to the economics and politics of production. This book seeks to reflect thatbalance between practical science, fundamental understanding and economics.

Citric acid derives its name from the Latin citrus, the citron tree, the fruit of whichresembles a lemon. The acid was first isolated from lemon juice in 1784 by Carl Scheele, aSwedish chemist (17421786), who made a number of discoveries important to the advanceof chemistry, amongst them hydrofluoric, tartaric, benzoic, arsenious, molybdic, lactic, citric,malic, oxalic, gallic and other acids as well as chlorine, oxygen (1772, published in Englishin 1780, predating the discovery by Priestly in 1774), glycerine and hydrogen sulphide.Citric acid was thus one amongst many natural organic acids.

Citric acid was produced commercially from Italian lemons from about 1826 in Englandby John and Edmund Sturge, but with the increasing importance of citric acid as an item ofcommerce, production was started in Italy by the lemon growers, who established a virtualmonopoly during the rest of the nineteenth century. Lemon juice remained the commercialsource of citric acid until 1919 when the first industrial process using Aspergillus nigerbegan in Belgium.

Lemon juice itself remains an important product. World lemon production averages about3.3 million metric tonnes (US Foreign Agricultural statistics); about 75 per cent comesfrom the United States, Italy, Spain and Argentina, with the rest from some 15 other producercountries.

Citric Acid Biotechnology2

Marketing of lemons is the subject of political control both in Europe and the USA. InEurope the processing of lemons to juice carries a processing subsidy which makes it attractiveto process the lemons rather than sell them as fresh produce; additionally the EU interventionmechanism results in significant quantities of lemons being destroyed. In the USA marketingis controlled by the United States Department of Agriculture (USDA) Lemon AdministrativeCommittee which determines how many lemons will be sold into the fresh market and whatgrowing areas will be allowed to sell them.

The economic result of any monopoly tends to be to make the product expensive; withoutthe spur of competition the control of costs, the development of the process and the efficiencyof production are neglected. Citric acid in the nineteenth century was no exception; theItalian monopoly resulted in high prices that tempted the entrepreneurs of the era to seekalternative sources of the increasingly useful product. Unable to find an alternative botanicalsource of citric acid, the nineteenth century advances in chemistry and microbiology wereexamined. By the turn of the century both possibilities existed.

1.2 Synthetic citric acid

Citric acid had been synthesized from glycerol by Grimoux and Adams (1880) and laterfrom symmetrical dichloroacetone (i) by treating with hydrogen cyanide and hydrochloricacid to give dichloroacetonic acid (ii), and converting this into dicyano-acetonic acid (iii)with potassium cyanide, which on hydrolysis yields citric acid (iv), as shown in Figure 1.1.

Several other routes using different starting materials have since been published. Allchemical methods have so far proved uncompetitive or unsuitable, mainly on economicgrounds, with the starting material worth more than the end product, although poor yieldsdue to the number of reaction steps in the synthesis and precautions necessary when handlinghazardous compounds involved have contributed to the problem.

1.3 Microbial citric acid

The concept of microbiological action yielding useful products followed from Pasteurspioneering studies on fermentation and resulted in systematic investigations of fungi andbacteria. Amongst them Wehmer, in 1893, showed that a Citromyces (now Penicillium)accumulated citric acid in a culture medium containing sugars and inorganic salts. Thiswork did not lead directly to a commercial process but the subsequent search for otherorganisms capable of this synthesis did. Many other organisms were found to accumulatecitric acid including strains of Aspergillus niger, A. awamori, A. fonsecaeus, A. luchensis, A.phoenicus, A. wentii, A. saitoi, A. lanosius, A. flavus, Absidia sp., Acremonium sp., Aschochyta

Figure 1.1 Synthesis of citric acid

A brief introduction to citric acid biotechnology 3

sp., Botrytis sp., Eupenicillium sp., Mucor piriformis, Penicillium janthinellum, P. restrictum,Talaromyces sp., Trichoderma viride and Ustulina vulgaris.

Currie (1917) found strains of A. niger that produced citric acid when cultured in mediawith low pH values, high sugar levels and mineral salts. Prior to this A. niger was known toproduce oxalic acid; the key difference was the low pH which, as we now know, suppressedboth the production of oxalic acid, which would be toxic, and gluconic acid, which has asignificantly higher production rate from sugar than citric acid. Currie subsequently joinedChas. Pfizer & Co. Inc. and his discovery formed the basis of the citric acid plant establishedin the USA by the firm in 1923. This plant and the other similar processes established firstin Belgium then in England by J.E.Sturge, in Czechoslovakia and in Germany in the nextfew years used the surface process. The details of this process are not well documenteddespite its long history, due in part to the restriction of information by manufacturers. Inbiotechnological terms, citric acid is known as a bulk, or low value, product. The market is,and always has been, very competitive, so the profit margins are small. Improvements inproductivity depend on the detail of the various processes, many of which are not easilyprotected by patents, so that secrecy is important and understandable.

1.4 Citric acid by the surface method

The general details of the original process are straightforward. The fungal mycelium isgrown as a surface mat on a liquid medium in a large number of shallow trays with acapacity of 50 to 100 litres. Each tray has a surface area of about 5 m2 and a depth ofbetween 5 and 20 cm. The trays are manufactured from high purity aluminium or stainlesssteel and usually can be lifted by just two men. The trays are stacked in racks in a chamberto allow operation under relatively aseptic conditions. Various sucrose sources were usedinitially but cane molasses and then beet molasses soon became the norm as the sugarsource. The molasses are diluted to the required concentration, usually 15 per cent andthe pH adjusted to 57. After sterilization, the medium is pumped into the trays andinoculation carried out directly from spores, either by adding a liquid suspension or byblowing the spores in with the air stream. Aerating the chambers is important for twopurposes, oxygenation and heat removal. The air requirement depends on the stage ofgrowth. Initially sterile air at low rates is used to prevent contamination during thegermination stage, which takes about 12 hours. Later, when growth is maximal, rates ofup to 10 m3 per cubic metre medium per minute are needed to ensure heat dispersal. Theheat generation is considerable, around 1 kJ h-1 m-3 medium and the surface and mediumtemperatures are ideally around 28C to 30C. This high volume air is not necessarilysterile, as contamination is normally not a problem once the pH has fallen, after about 24hours growth. The pH falls to about 2, or slightly lower, and remains at that level until theend of the process, hence the need for high-grade materials for the construction of thetrays. The incoming air is humidified to 4060 per cent to prevent moisture loss from thehigh surface area of the medium. Cultivation continues for 8 to 15 days, with the objectiveof minimizing the residence time to maximize the plant productivity. The details of time,productivity and yield are closely guarded secrets, but productivity of the order of 1 kgper square metre per day can be obtained and yield is up to 75 per cent of the initial sugarlevel. At the end of the process, which can be monitored by total acid production orjudged by experience, the mycelial mat is removed by filtration and washed, as it containsup to 15 per cent of the total citric acid. The washings and spent medium are treated withlime (calcium hydroxide) at about 90C to precipitate the insoluble tri-calcium tetrahydrate


salt of citric acid. It is not possible to crystallize the acid directly from the crude molassesmedium although this can be done if pure sucrose is used as the carbon source. Theprecipitate of calcium citrate is washed and suspended in enough sulphuric acid toprecipitate the calcium as calcium sulphate. This releases the citric acid into solutionfrom where it can be treated further as required.

The surface process, though commercially profitable for many years, is labour intensiveand inefficient in its use of space; there is a limit as to how high a large tray can be lifted!The production of citric acid by surface culture was challenged at the beginning of the1940s by the development of submerged fermentation processes. When Shu and Johnsonpublished their work on the effect of medium ingredients and their concentrations oncitric acid production in submerged culture, the fundamental technology for submergedproduction was ready to be exploited on an industrial scale (Shu and Johnson, 1948a,1948b).

1.5 The submerged process for production of citric acid

The submerged process has become the method of choice in the industrialized countriesbecause it is less labour intensive, gives a higher production rate, and uses less space. Severaldesigns of reactor have been used, particularly in pilot scale systems; the stirred tank reactoris the most common design although air-lift reactors, with a higher aspect ratio than thestirred tank reactor are also used. The reactors are constructed of high-grade stainless steel,an important requirement in view of the low pH levels developed, the ability of citric acid tosolubilize metal ions and the presence of manganese in stainless steels. Inferior grades ofsteel have caused problems in the past, both of leaching and pitting or general corrosion.Industrial rumours suggest it may still happen though not by design! The empirical processof conditioning a reactor, whereby a few batches are processed before optimal productionlevels are achieved, may be related to this problem.

The other general requirement for reactors for citric acid production is the provision ofaeration systems that can maintain a high dissolved oxygen level. With both tank and towerreactors sterile air is sparged from the base, although extra inputs are often used with towerreactors. The reactor may be held above atmospheric pressure to increase the rate of oxygentransfer into the fermentation broth. The influence of dissolved oxygen on citric acidformation has been examined and the dissolved oxygen levels are routinely monitored. Theoxygen levels are also affected by the rheology of the broth.

A typical plant will consist of four areas: medium preparation, reactor section, brothseparation and product recovery. The medium preparation will involve dilution of themolasses, or other raw material, addition of nutrients and other pre-treatment such asferrocyanide, and sterilization, either in-line or in the reactor. Where in-line sterilization isused the reactors are steam sterilized separately. It is usual to prepare an inoculum for theproduction reactor in a smaller reactor, in which the conditions may be modified to giverapid growth rather than product formation. Primary inoculation is by spores and the initialphase of the growth is critical.

When a separate inoculum stage is used, the correct stage for transfer, characteristicallybetween 18 and 30 hours, is judged by pH level. Production temperature, like the inoculumtemperature, is about 30C. The process is allowed to continue until the rate of citric acidproduction falls below a predetermined value, which is reached many hours before theproduction ceases altogether.


Many reports suggest that the morphology of the mycelium is crucial to the ultimateyield; not only with respect to the shape of hyphae, but also their aggregation. Severalstudies suggest that hyphae should be abnormally short, bulbous and heavily branched. It isrecognized that this condition is brought about by manganese deficiency or related to theaddition of ferrocyanide, which is probably the same thing. The mycelium should also formsmall (less than 0.5 mm) pellets with a smooth, hard surface. Such pellets are producedwhen a number of factors are controlled, such as ferrocyanide levels, manganese levels, lowiron (less than 1 ppm), low pH, control of aeration and agitation or the amount of sporeinoculum.

It is clear that this morphological appearance is not in itself necessary for a successfulyield, but is a result of the correct process parameters. Pellet formation is not necessary, butdoes give a broth with a lower energy requirement for mixing. When a change to a filamentousgrowth type occurs, the dissolved oxygen level may fall by 50 per cent for a fixed input.That filamentous growth can give satisfactory yields has been demonstrated and considerationof the diffusion characteristics of pellets versus filamentous mycelium would suggest thatwhile yields may be similar, productivity should be greater without the additional diffusionalconstraint of pellets.

Aeration is a significant factor in the cost of the process, and although a constant aerationrate is used in many laboratory scale studies, the industrial practice is to use relatively lowaeration rates initially (0.1 vvm) rising to 0.51 vvm as growth proceeds. Such aerationrates will lead to foaming and various devices and agents are available to minimize theproblem. Although very high yields are possible, the productivity is a more importantconsideration on an industrial basis, and it is rare that the process is allowed to continue tothe maximum yield.

The processes run today owes much to the pioneering work carried out by D.S.Clarkand his co-workers at the Northern Regional Research Laboratories in Canada during the1950s and early 1960s. Here, the technology for large-scale production of citric acid withA. niger using molasses was established. After the fermentation characteristics were workedout, attention was given to the controlling mechanisms of the fermentation. Numerousreports have been published on the role of metal ions on the citric acid cycle, in particular.After decades of academic discussion, there is general agreement about the factors thatregulate the fermentation and give rise to the high yields obtained in industry (Mattey,1992).

1.6 Continuous and immobilized processes

A process for continuous production of citric acid has been described (Kristiansen andSinclair, 1979), but no commercial application of this has been made in spite of the highproductivity values obtained (Kristiansen and Charley, 1981). The process does not use thecarbon source as the limiting substrate so that excess sugar will pass out of the reactor. Asthe carbohydrate substrate is one of the major cost factors, the continuous process will beless efficient than the batch process. This might be overcome by using several reactors inseries, but this offsets any advantage from the continuous process.

Fed-batch processes have been used industrially so that the conversion of sugarconcentrations greater than 15 per cent can be achieved, but the gain does not seem to besufficient to allow the fed-batch method to become standard.

The possibility of using the mycelium in an immobilized system has occurred toseveral workers and attempts on a small scale have been reported. Immobilization of


mycelium in alginate beads or collagen proved possible, but with very low productionrates. The difficulties of avoiding oxygen limitation when preparing beads, andpreventing further growth, which reduces oxygen transfer rates, have led to theimmobilization of conidia which are then grown under nitrogen limitation to the desiredcompact pellet. While giving a manageable system, the productivity was still too low tobe of industrial interest.

Other constructs for immobilization that have been more successful are the use of exchangefiltration, and a rotating disc with an adhering mycelial film, reminiscent of sewage treatmenttechniques. These radical methods are unlikely to gain acceptance, even were they to giveeconomic productivity gains, unless the engineering problems of scale-up can be overcomewithout making the capital costs too large.

1.7 Yeast based processes

From about 1965 methods using yeasts were developed, first from carbohydrate sources,then from n-alkanes. At this time hydrocarbons were relatively cheap and plants werebuilt to use the method. The economics have altered since then and plants that have beenbuilt to utilize both yeast technologies have apparently switched back to carbohydratefeedstocks.

The potential advantages of using yeasts rather than filamentous fungi are the higherinitial sugar concentrations that can be tolerated and the faster conversion rates possible.Further, the insensitivity to metal ions means that crude (and hence cheaper) grade molassescan be used without costly pre-treatment. Since 1968, when the patent for citric acidproduction from molasses by eight genera of yeasts was allowed, there have been manyprocess modifications reported. Candida, Hansenula, Pichia, Debaromyces, Torulopsis,Kloekera, Trichosporon, Torula, Rhodotorula, Sporobolomyces, Endomyces, Nocardia,Nematospora, Saccharomyces, and Zygosaccharomyces species are known to producecitric acid from various carbon sources. Out of these genera the Candida species, includingC. lipolytica, C. tropicalis, C. guillermondii, C. oleophila and C. intermedia have beenused.

The original process incorporated calcium carbonate into the medium to maintain a neutralpH, and generally a pH above 5.5 was used. Various additions have been proposed to reducethe isocitric acid contamination that afflicts yeasts even on carbohydrate media. Halogensubstituted alkanoic mono- or di-substituted acids, n-hexadecyl citric acid or trans-aconiticacid, and even lead acetate have been patented, despite the possibility of toxic residues inthe resulting citric acid. Many mutants have been selected for reduced isocitrate production.An osmophilic strain, which would convert sugar concentrations as high as 28 per centwithout pre-treatment of the molasses substrate, has been patented.

Tower reactors of fairly standard design are used, but with improved cooling systems asthe rate of heat production is high. A continuous process has been described where the pHis maintained at 3.5 with ammonium hydroxide.

The industrial production of citric acid from n-alkanes is not now economic, although aplant was built, and operated, around 1970 at Saline, Reggio Calabria, Italy (Liquichimica).This process was based on a low aconitase mutant of C. lipolytica in a batch process withstirred, aerated tank reactors of 400 m3, operating on a 72 hour cycle. The conversion fromalkanes was reported to exceed 130 per cent (by weight). The theoretical yield is 250 percent, but part of the alkanes was converted to biomass and carbon dioxide. The yeast wasremoved by centrifugation and the purification was traditional. The medium used was based


on the process developed for the yeast strain that had a substrate concentration of 10 percent n-decane, although n-alkanes from 9 to 20 carbons could be used. The availability andcost of Libyan n-alkanes, which lead to the development of this and other plants, includingthe dual substrate plants, has changed over the last three decades. One unique feature of then-alkane process is the insolubility of the substrate. To ensure a rapid conversion the n-alkane has to be thoroughly dispersed, so additives such as polyoxypropylene glycol ether,at concentrations from 20 to 200 ppm, are used to enhance this.

1.8 The koji process

A third method for the production of citric acid is the koji process, using Aspergillus species.This is the solid state equivalent of the surface process described previously. It was originallydeveloped in Japan where it uses the readily available rice bran and fruit wastes. It is confinedto south-east Asia and is a relatively small-scale process. The carbohydrate source, which isprincipally starch and cellulose, is sterilized by steaming and the resulting semi-solid paste(about 70 per cent water), at a pH of about 5.5, is inoculated by spraying on spores of A.niger. Additions of ferrocyanide or copper may be made. The incubation temperature is30C and the process takes about four to five days. Yields are low because of the difficultyof controlling trace metals and the process parameters. The fungus produces sufficientcellulases and amylases to break down the substrate, though the low yields may reflect therate limitations of this step.

1.9 Uses of citric acid

Citric acid is used in food, confectionery and beverages, in pharmaceuticals and inindustrial fields. Its uses depend on three properties: acidity, flavour, and salt formation.Chemically citric acid is 2-hydroxy-1,2,3-propane tricarboxylic acid (77-92-9). It hasthree pKa values at pH 3.1, 4.7 and 6.4. As these three values are relatively close togetherthe second H+ is appreciably dissociated before the first is completed, and similarly withthe third. Because of this overlapping the solution is well buffered throughout the titrationcurve and there are no breaks from about pH 2 (the approximate pH of a 0.2M solution)to pH 7.

Citric acid forms a wide range of metallic salts including complexes with copper, iron,manganese, magnesium and calcium. These salts are the reason for its use as a sequesteringagent in industrial processes and as an anticoagulant blood preservative. It is also the basisof its antioxidant properties in fats and oils where it reduces metal-catalysed oxidation bychelating traces of metals such as iron. There are two components to its use as a flavouring:the first is due to its acidity, which has little aftertaste; the second to its ability to enhanceother flavours.

A process to remove sulphur dioxide from flue gases has been developed where citricacid is used as a scrubber, forming a complex ion which then reacts with H2S to give elementalsulphur, regenerating citrate. This may become more important with increased environmentalpressures.

Citric acid esters of a range of alcohols are known; the triethyl, butyl and acetyltributylesters are used as plasticizers in plastic films and monostyryl citrate is used instead of citric acidas an antioxidant in oils and fats. A summary of the uses of citric acid is given in Table 1.1.


1.10 Effluent disposal

Regardless of the method of production the disposal of waste is an increasing problemfor manufacturers both from a cost and a regulatory viewpoint. Gypsum (calciumsulphate) is not valuable enough to purify and use in, for example, plaster. It may bedisposed of to landfill sites, at a cost, and in some cases may be pumped out to sea,where tidal conditions permit. A more serious problem is the disposal of the filtratefrom the precipitation where molasses has been used as a raw material; the waste isnon-toxic, but has a high biological oxygen demand, so that it cannot be disposed of torivers untreated. Anaerobic digestion, with fuel gas as a useful by-product, is probablythe future method of choice, although animal feedstuff formulation in the form ofcondensed molasses solubles is another possibility. It can also be used as a medium forthe growth of yeasts for animal feeds.

1.11 Conclusions

Books must follow science, not science books.(Francis Bacon, Propositions touching Amendment of Laws)

For the last 80 years citric acid has been produced on an industrial scale by the fermentationof carbohydrates, initially exclusively by Aspergillus niger, but in recent times by Candidayeasts as well, with the proportion derived from the Candida process increasing. The higherproductivity of the yeast-based process suggests it will be the method of choice for any newplants that may be built.

The intimate knowledge about the large-scale fermentation and subsequent recoveryprocesses are still regarded as industrial property. Nevertheless, the citric acid process is

Table 1.1 Applications of citric acid


one of the rare examples of industrial fermentation technology where academic discoverieshave worked in tandem with industrial know-how, in spite of an apparent lack of collaboration,to give rise to a very efficient fermentation process.

The current world market for citric acid and its derivatives is difficult to estimateaccurately; no international statistics are collected, but industry estimates suggest that upwardsof 400 000 tonnes per year may be produced. Citric acid is a mature product but theupward trend in its use seen over many years is an annual 23 per cent increase.

The price is such that profit margins are low, and with significant, but erratic, quantitiesappearing on the world market from countries such as China the situation is unlikely toimprove.

The lemon, which started it all, is doing well, with an estimated world production of 3 to4 million tonnes per year. Commercial varieties such as Eureka are all high acid lemons,with the acid content exceeding 4.5 per cent by weight, so that some 140 000 tonnes ofcitric acid are still produced by lemons!

The various themes touched on in this introduction are dealt with in greater depth in thefollowing chapters.

1.12 References

COOPER, W C and CHAPOT, H, 1977. Fruit Productionwith special emphasis on fruit for processing.In Citrus Science and Technology, Vol. 2. Eds S Nagy, P E Shaw and M K Veldhuis (AVI PublishingCo., Westport, CT, USA).

CURRIE, J N, 1917. The citric acid fermentation of A. niger, Journal of Biological Chemistry, 31, 5.GRIMOUX, E and ADAMS, P, 1880. Synthese de lacide citrique, C.R.Hebd. Seances Acad. Sci., 90,

1252.KRISTIANSEN, B and CHARLEY, R C, 1981. The effect of medium composition on citric acid

production in continuous culture, Presented at 2nd European Congress of Biotechnology, UK.KRISTIANSEN, B and SINCLAIR, C G, 1979. Production of citric acid in continuous culture,

Biotechnology and Bioengineering, 21, 297.MATTEY, M, 1992. The production of organic acids, CRC Critical Reviews in Biotechnology, 12, 81.PASTEUR, L., 1875. Nouvelle observations sur la nature de la fermentation alcoolique, C.R. Acad.

Sci., 80, 452.REUTHER, W, CALAVAN, E C and CARMAN, G E, 1967. The Citrus Industry, Vol. 1. History,

World Distribution, Botany and Varieties. Univ. Calif. Div. Agric. Nat. Res., San Pablo, California.ROSENBAUM, J B, MCKINNEY, W A, BEARD, H L, CROCKER, L and NISSEN, W I, 1973.

Sulphur Dioxide Emission Control by Hydrogen Sulphide Reaction in Aqueous Solution. TheCitrate System. US Bureau of Mines, Report 1774.

SCHEELE, C, 1793. Crells Ann. 2, 1 1784, from Smmtliche Physische und Chemische Werke.Hermbstdt (Berlin).

SHU, P and JOHNSON, M J, 1948a. Citric acid production submerged fermentation with Aspergillusniger, Industrial and Engineering Chemistry, 40, 1202.

SHU, P and JOHNSON, M J, 1948b. The interdependence of medium constituents in citric acidproduction by submerged fermentation, Journal of Bacteriology, 54, 161.

WEHMER, C, 1893. Note sur la fermentation Citrique, Bull. Soc. Chem. Fr, 9, 728.

11

2

Biochemistry of Citric AcidAccumulation by Aspergillus niger

MARKUS F.WOLSCHEK AND CHRISTIAN P.KUBICEK

2.1 Introduction

The biochemical mechanism by which Aspergillus niger accumulates citric acid hasattracted the interest of researchers since the late 1930s when the optimization of thisaccumulation to give a commercial process began. In this sense, the various theorieswhich have been proposed to explain the accumulation of citric acid in such high yieldsalso reflect the general biochemical knowledge at the time the respective research wasdone. In view of the high input into this research through more than 50 years it is thereforerather disappointing that there is still no explanation of the biochemical basis of thisprocess which would consistently explain all the observed factors influencing thisfermentation. Reasons for this are manifold. First, citric acid is only accumulated whenseveral nutrient factors are present, either in excess (i.e. sugar concentration, H+, dissolvedoxygen), or at suboptimal levels (trace metals, nitrogen and phosphate), and thus is subjectto multifactorial influence. Hence it is unlikely that single biochemical events are solelyresponsible for citric acid overflow.

Secondly, an appreciable part of the literature consists of work which has been performedusing low or only moderately producing strains or by applying nutrient conditions not optimalfor citric acid production, and while this may be justified for special reasons in individualcases, the respective results are not comparable to those obtained by others. Moreover, theirsignificance for the understanding of the commercial citric acid fermentation is questionable.

Thirdly, the biochemical knowledge of filamentous fungi is still significantly inferior tothat of, for example, Saccharomyces cerevisiae or higher eukaryotes and, moreover, resultsfrom these sources cannot be uncritically transformed to filamentous fungi, which impedesa biochemically correct interpretation of results in several areas. Hence, although aconsiderable amount of basic biochemical research has been carried out with A. niger, thepresent state of understanding of the events relevant for citric acid accumulation (not to sayproduction) is still a poorly resolved puzzle.

This chapter attempts to draw the currently recognizable picture and to aid in the furtherfitting together of the other scattered bits and pieces.


2.2 Glucose catabolism in A. niger and its regulation

2.2.1 The citric acid biosynthetic pathway

It is well known, since the famous tracer studies by Cleland and Johnson (1954), and Martinand Wilson (1951), that citric acid is mainly formed via the reactions of the glycolyticpathway. Like most other fungi Aspergillus spp. utilize glucose and other carbohydrates forenergy and cell synthesis by channelling glucose into the reactions of the glycolytic and thepentose phosphate pathway, respectively. The pentose phosphate pathway accounts for onlya minor fraction of metabolized carbon during citric acid fermentation, and this decreasesthroughout prolonged cultivation (Legisa and Mattey, 1986; Kubicek, unpublished data).Legisa and Mattey (1988) speculated that this may be due to inhibition of 6-phosphogluconatedehydrogenase by citrate, but evidence for this is lacking. It should be noted that botharabitol and erythritol are accumulated as by-products until late stages of the fermentation(Roehr et al., 1987); hence a complete blockage of the pentose phosphate pathway isobviously not taking place.

A. niger possesses a further pathway of glucose catabolism which is catalyzed by glucoseoxidase (Hayashi and Nakamura, 1981). This enzyme is induced by high concentrations ofglucose and strong aeration in the presence of low concentrations of other nutrients (Mischaket al., 1985; Rogalski et al., 1988; Dronawat et al., 1995), conditions which are also typicalfor citric acid fermentation; glucose oxidase will hence inevitably be formed during thestarting phase of citric acid fermentation and convert a significant amount of glucose intogluconic acid. However, due to the extracellular location of the enzyme, it is directlyinfluenced by the external pH and will be inactivated at pH

Biochemistry of citric acid accumulation by A. niger 13

dioxide and oxygen in the exit air of a pilot plant citric acid fermentation, observed thatduring the first 70 hours of fermentation the respiratory coefficient (i.e. CO2 released/O2taken up) is close to 1; it starts to decrease thereafter and reaches the level predicted fromthe operation of the pyruvate carboxylase reaction (0.66) only at stages where citrateaccumulation is already taking place at a constant rate (e.g.

Figure 2.2 Metabolic and regulatory network of citric acid biosynthesis from sucrose in A.niger. For convenience, sucrose is assumed to be split into glucose and fructose by invertaseextracellularly (Boddy et al., 1993; Rubio and Maldonado, 1995) and only themonosaccharides are taken up. The double line indicates the plasma membrane, the hatcheddouble line the mitochondrial membrane. Circles inserted into the membranes indicateknown or assumed transport steps (hatched: characterized in A. niger; full: assumed, but notyet characterized; empty: countertransport, to be verified). Thick lines and arrows indicatemetabolic reactions; thin lines and arrows indicate regulatory interactions (*activation: //inhibition). Intermediates of regulatory importance are boxed.


2.2.2 Transcriptional regulation of the citric acid synthesizing pathway

It is uncertain to what extent the apparently high flux through the glycolytic pathway, whichis obviously necessary for citric acid accumulation, requires an activation of transcriptionof the genes encoding glycolytic and other enzymes (e.g. citrate synthase). The quantificationof enzyme activities in cell-free extracts of A. niger mutants, which were selected accordingto a reduced lag in growth on high sucrose concentrations and correspondingly increasedrates of citric acid accumulation, revealed enhanced hexokinase and phosphofructokinaseactivities (Schreferl-Kunar et al., 1989). Also, a class of A. niger mutants, resistant to 2-desoxyglucose and displaying reduced hexokinase activity, exhibited decreased rates ofcitric acid production (Fiedurek et al., 1988; Kirimura et al., 1992; Steinbck et al., 1994).Torres et al. (1996a) showed that high glucose concentrations (>50 g/l) are a prerequisitefor the formation of a low-affinity glucose transporter. However, knowledge of thetranscriptional regulation of the respective genes is still lacking.

Only preliminary data are as yet available to understand whether an enhancement oftranscription of selected glycolytic genes would increase the rate of citric acid accumulation.Ruijter et al. (1996b) haveselectively and in combinationamplified the genes encodingphosphofructokinase 1 (pfkA) and pyruvate kinase (pkiA), but the rates of citrate accumulationby the moderately citric acid producing strain used (N400) were not increased. Torres (1994a,1994b), using the biochemical system theory and a constrained linear optimization method,calculated that the activities (Vmax) of at least seven glycolytic enzymes must besimultaneously increased to obtain an effect. Clearly, such an increase can only be achievedby appropriate manipulation of the transcription factors regulating the genes encoding theenzymes for citric acid biosynthesis.

Unfortunately, transcriptional regulation of glycolytic genes has not yet been studied insufficient detail in A. niger nor in any related fungus. In Saccharomyces cerevisiae, mutationsin the GCR1 gene, which encodes a DNA-binding protein (Baker, 1986, 1991), were foundto exhibit strongly reduced levels of most glycolytic enzymes. Another protein, GCR2, wasshown to interact physically with GCR1 (Uemura and Jigami, 1992). The authors proposedthat both factors co-operate together in a transcriptional activation complex. Further factorsinvolved in the regulation of the glycolytic genes have been described in yeast (RAP1,REB1, ABF1; Brindle et al., 1990; Chambers et al., 1990; McNeil et al., 1990; Huie et al.,1992). GCR1-binding sites are generally located near RAP1-binding sites (Huie et al., 1992).Furthermore, several glycolytic genes contain consensus binding sites for binding of ABF1and REB1 in the vicinity of RAP1- and GCR1-binding sites (Brindle et al., 1990; Chamberset al., 1990; Chasman et al., 1990; Scott and Baker, 1993).

Figure 2.3 Pathway of oxalate biosynthesis by Aspergillus niger. Note that concentrations ofacetate corresponding to those of oxalate have not been detected in culture filtrates of A.niger, and the metabolism of acetate therefore requires further study


It is intriguing that the above named binding sites have so far not been detected in the 5'-noncoding sequences of the few glycolytic genes studied in A. niger or the close relativeAspergillus nidulans (Table 2.1, Figure 2.4). Transcriptional regulation of glyceraldehyde-3-phosphate dehydrogenase (Punt et al., 1988, 1990, 1992) and of 3-phosphoglycerate kinase(Clements and Roberts, 1986; Streatfield et al., 1992) has been studied in some detail in theclosely related fungus A. nidulans. Its transcription depends on positive control by severalco-operating DNA-binding proteins since a truncated core promoter of the pgkA gene onlycontaining the CAAT, TATA and CT-rich elements could not trigger transcription. Punt etal. (1988) identified a glycolytic box as responsible for transcription. No differences inexpression of gpdA were observed on 1% glucose or 0.1% fructose (Punt et al., 1990).

A 24-bp region, which shares 60 per cent similarity with the glycolytic box, is alsopresent at -638 and -488 of the pgkA promoter (Figure 2.4). However, another sequence,located between -161 and -120, in the pgkA promoter was shown to be essential for expressionof the respective gene. It consists of two non-overlapping octameric sequences that matchin seven out of eight nucleotides to the higher eukaryotic consensus ATGCAAAT (Falkneret al., 1986).

A 17-base pair sequence was found in the 5'-regions of the A. nidulans and A. niger pkiAgenes that may act as an upstream regulating sequence (de Graaff et al., 1992). This sequencewas shown to be distinct from the proposed cis-acting element mediating increasedtranscription of pyruvate kinase on glycolytic carbon sources (de Graaff et al., 1988).

Table 2.1 Genes encoding enzymes involved in the biosynthesis of citric acid by A. niger,which have already been cloned from A. niger or other Aspergillus spp.

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2.2.3 Glucose metabolism and its regulation

The disappointing result that amplification of selected genes did not lead to an increase inthe rate of citric acid accumulation by A. niger indicates the operation of very tight finecontrol of at least some of the enzymes involved. In fact, this fine control was already amajor target of investigation throughout the early 1980s, and as a consequence several ofthe enzymes involved in it have been comparably well characterized (Table 2.2). Mostrecently, the method to study the concentration of intracellular metabolites in A. niger hasalso been critically reassessed (Ruijter and Visser, 1996). Based on these data, Figure 2.2shows those regulatory interactions between metabolites and enzymes which are believedto be of major importance to the regulation of citric acid biosynthesis in A. niger. Similar tothe situation in yeast and higher eukaryotes, citrate and phosphoenolpyruvate (PEP) seemto be the major factors negatively affecting the glycolytic flux, whereas Fructose-2,6-diphosphate (Fru-2,6-P2) and Fru-1,6-P2 appear to be the major activators.

2.3 Regulation of citric acid biosynthesis

Citrate is one of the best known inhibitors of glycolysis, and the ability of A. niger tooverproduce citrate by an active glycolytic pathway has therefore attracted biochemicalinterest for a long time; it is considered to be of major consequence for the fermentation rate(cf. Habison et al., 1979). However, under appropriate nutrient conditions (see below), thisinhibition is more than counteracted by the accumulation of various positive effectors ofPFK1 (NH4+, inorganic phosphate, AMP, Fru-2,6-P2), and hence this feedback does notoccur (Habison et al., 1983; Arts et al., 1987).

A series of investigations by Kubicek and co-workers favour the assumption that Fru2,6-P2 may play a major role in the counteraction of citrate inhibition: Kubicek-Pranz et al.(1990) found that the triggering of citric acid accumulation by replacing A. niger in highconcentrations (14% w/v) of sucrose or glucose (Shu and Johnson, 1948b; Xu et al.,1989a) is paralleled by a rise in the intracellular concentration of Fru-2,6-P2. Also, myceliacultivated on carbon sources which allow higher yields of citric acid (i.e. those which aretaken up rapidly; Hossain et al., 1984; Kubicek and Roehr, 1986; Honecker et al., 1989;Xu et al., 1989a) showed higher concentrations of Fru-2,6-P2. The concentration of Fru-2,6-P2 correlates therefore positively with the rate of citrate production, and this fact maybe responsible for the lack of citrate inhibition of PFK1. The reason for the increased F-2,6-P2 level is not completely clear, but it appears to be due to an increased Fru-6-Psupply for PFK2, since this enzyme is only poorly regulated in A. niger (Harmsen et al.,1992).

The biosynthesis and regulation of Fru-2,6-P2 links regulation of PFK1 to that of earliersteps in glycolysis. Torres (1994a, 1994b) has recently concluded from theoreticalcalculations that a major part of the actual control of citric acid production must occur athexose uptake and/or phosphorylation, which is in accordance with such an assumption.The biochemistry of these early steps in A. niger glycolysis is not completely clear, howeverSteinbck et al. (1994) found a single hexo/glucokinase only in the citric acid producingstrain ATCC 11414, which was inhibited by citrate and weakly sensitive to trehalose-6-phosphate (Arisan-Atac et al., 1996). The inhibition by citric acid was due to chelation ofMg2+ which is required to chelate the co-substrate ATP, and is most probably irrelevantunder physiological conditions where Mg2+ is present in excess. However, the inhibition bytrehalose-6-phosphate appears to be relevant to the flux towards citric acid, since a


recombinant strain of A. niger, which carries a disrupted copy of the constitutively expressedtrehalose-6-phosphate synthase gene tpsA (Wolschek and Kubicek, 1997), produces citricacid at increased rates (Arisan-Atac et al., 1996). Similarly, a strain bearing multiple copiesof tpsA and hence overproducing trehalose-6-phosphate synthase exhibited a reduced rateof citrate production. These data indicate that the cellular level of trehalose-6-phosphateregulates the flux from glucose to citric acid and are thus in accordance with the conclusionsof Torres et al. (1996a) that hexokinase most likely accounts for the major part of regulationat the early steps of glycolysis, thereby supplying an increased concentration of substratefor PFK2.

However, most recently Panneman et al. (1996) reported on the isolation andcharacterization of a glucokinase from A. niger N400, a strain producing only low levels ofcitric acid, which has properties different from the hexo/glucokinase purified by Steinbcket al. (1994). They also concluded that by analogy with A. nidulans (Ruijter et al., 1996a)there may be also at least one separate hexokinase as well. The difference between theresults of Steinbck et al. (1994) and Panneman et al. (1996) are currently unresolved.Hybridization of an A. niger ATCC 11414 DNA with a Kluyveromyces lactis hexokinase-encoding gene as a probe showed hybridization to a single fragment only (F.Narendja andC.P.Kubicek, unpublished data). The gene from strain ATCC 11414 has recently been clonedin our laboratory, and its characterization has to be awaited for clarification of this situation.Whatever the results of this investigation, the results by Arisan-Atac et al. (1996) clearlyshow that a relief from trehalose-6-phosphate inhibition positively influences the glycolyticflux at high sugar concentrations, and the hexose-phosphorylating step is therefore a majorregulatory point in this fermentation.

Glucose uptake by A. niger was investigated by Torres et al. (1996a). A. niger ATCC11414 contains two transporters with different Km and Vmax. However, the high-affinitypermease can only be detected during growth on low glucose concentration (1% w/v),whereas the low-affinity permease is detectable in the presence of high glucoseconcentrations. The latter may therefore contribute to the increased glycolytic flux duringgrowth on high glucose concentrations.

Several lines of evidence suggest that the regulation of PFK1 by Fru-2,6-P2 may not bethe only parameter regulating citrate accumulation. Citrate inhibition of PFK1 also seemsin vivo to be antagonized by ammonium ions (Habison et al., 1979). This antagonism isfunctionally linked to the well known effect of trace metal ions (particularly manganeseions) on citric acid accumulation (Shu and Johnson, 1948b; Tomlinson et al., 1950; Trumpyand Millis, 1963), as one of the effects caused by manganese deficiency is an impairment ofmacromolecular synthesis in A. niger (Kubicek et al., 1979a; Hockertz et al., 1987), whichcauses increased protein degradation (Kubicek et al., 1979a; Ma et al., 1985). As aconsequence, mycelia accumulate elevated concentrations of NH4+ (Kubicek et al., 1979a).Proof for the role of manganese ions in this process has been obtained by the isolation ofmutants of A. niger whose PFK1 was partially citrate-insensitive and whose citric acidaccumulation was simultaneously more tolerant to the presence of Mn2+ (Schreferl et al.,1986). Furthermore, several authors have reported that the exogenous addition of NH4+during citric acid fermentation even stimulates the rate of citrate production (Shepard, 1963;Choe and Yoo, 1991; Yigitoglu and McNeil, 1992), which is consistent with this effect ofNH4+ on PFK1. The latter authors documented that both the time of addition as well as theconcentration of NH4+ were important, and its addition during inappropriate fermentationphases even decreased acid accumulation.

The reason for the impairment of macromolecular synthesis under manganese deficientcultivation conditions had originally been assumed to be at the translational level (Ma et


al., 1985). However, Hockertz et al. (1987) have demonstrated that the absence ofmanganese ions from the nutrient medium of A. niger causes a reversible inhibition ofDNA, but not RNA biosynthesis. This is supported by the findings that the effect ofmanganese deficiency can be mimicked by addition of hydroxyurea, an inhibitor ofribonucleotide reductase (Hockertz et al., 1987). They proposed that manganese deficiencymay primarily impair DNA synthesis by causing a shortage of desoxyribonucleotidesrequired for DNA replication.

A further mechanism of regulation of PFK1 was proposed by Legisa and co-workers,who postulated that PFK1 is regulated by phosphorylation by cyclic-AMP dependent proteinkinase A (Legisa and Bencina, 1994). They speculate that a high concentration of sucrosecauses an increase in mycelial cyclic-AMP levels which trigger the phosphorylation ofPFK1, thereby converting an inactive (non-phosphorylated) form into an active(phosphorylated) form (Legisa and Gradisnik-Grapulin, 1995). The support for their modelis their observation that PFK1 was inactivated by treatment with alkaline phosphatase (Legisaand Bencina, 1994). However, this model, while intriguing, has to be treated cautiouslyuntil solid evidence for it has been obtained, as the molecular weight of the PFK1 purifiedby Legisa and Bencina (1994) and used for their studies was 48 kDa which is not that ofnative PFK1 (84 kDa). Moreover, the method section of their paper does not indicate whether(and how) the alkaline phosphatase had been removed or inactivated prior to the PFK1assay. If this was not done, the inactivation of PFK1 may have been due to a removal ofFru-6-P from the assay and thus be an artefact. Proof for a regulation of PFK1 byphosphorylation is therefore still needed.

A stimulation of citric acid accumulation by increased cyclic-AMP levels had also beenpostulated earlier (Wold and Suzuki, 1973, 1976a, 1976b). They showed that the stimulatoryeffect was dependent on the zinc concentration of the medium. Adenylate cyclase from A.niger has been described as Zn2+ dependent (Wold and Suzuki, 1974). A bottleneck of theirinvestigations, however, is that they were using 1% (w/v) sucrose throughout, and hence therelevance of their findings to the effect of zinc under citric acid fermentation conditions isunclear. Xu et al. (1989b) studied the intracellular concentration of cyclic-AMP in A. nigerduring citric acid biosynthesis on media with and without Mn2+ ions added, and with high(14%) and low (1%) sucrose concentrations. They reported that the cyclic-AMP levels weregrowth rate dependent, and comparable if phases of similar growth rates were compared.Whether or not cyclic-AMP is in fact involved in the regulation of citrate overproductionremains to be assessed.

2.4 Role of citrate breakdown in citrate accumulation

2.4.1 Role of the citric acid cycle

The reason why A. niger accumulates such massive amounts of citric acid has, since theearly studies by Ramakrishnan et al. (1955), attracted numerous investigations. Althoughcitrate has been considered an overflow product (Foster, 1949), which implies that itaccumulates as a result of an excessive substrate supply rather than a limited catabolism,an excessive amount of work has been concerned with the attempt to identify a bottleneckin the tricarboxylic acid cycle as the reason for its accumulation. Numerous workersclaimed that inactivation of an enzyme degrading citrate (e.g. aconitase or the isocitratedehydrogenases) would be essential for the accumulation of citric acid (for review seeSmith et al., 1974; Berry et al., 1977; Roehr et al., 1983, 1996; Kubicek and Roehr,


1986). While this view has an extraordinary long half-life in the review literature, solidevidence for the presence of an intact citric acid cycle during citric acid fermentation waspresented 25 years ago (Ahmed et al., 1972), and explanations based on this view aretherefore simply incorrect.

The requirement of citric acid accumulation of a deficiency in some metal ions (e.g.Mn2+, Fe3+) has frequently been used to explain an inhibition of some enzymes of the TCAcycle (for review see Kubicek and Roehr, 1986). Thus, iron deficiency has been claimed toinhibit aconitase (Szczodrak and Ilczuk, 1985). However, the activity of this enzyme duringcitric acid accumulation has been demonstrated clearly by others both in vitro (La Nauze,1966; Ahmed et al., 1972; Mattey, 1977) as well as in vivo (Kubicek and Roehr, 1985). Itshould be kept in mind that the enzymes of the respiratory chain, which also require iron,are highly active during citric acid accumulation (Ahmed et al., 1972; Hussain et al., 1978).By a similar rationale, the necessity for Mn2+ deficiency has been used to claim an inhibitionof either of the two isocitrate dehydrogenases which require divalent metal ions for activity(cf. Gupta and Sharma, 1995). However, this requirement is for chelation of the substrate(i.e. isocitrate; cf. Bowes and Mattey, 1979; Meixner-Monori et al., 1986). In view of thefact that Mg2+ (which is present in excess) can take over the chelating role of Mn2+ efficiently,this interpretation is unlikely to explain the effect of Mn2+.

Several other explanations for citric acid accumulation are based on the postulation of ametabolic inhibition of the NADP-specific isocitrate dehydrogenase by citrate (Mattey, 1977)or glycerol (Legisa and Mattey, 1986), which would create a bottleneck in the tricarboxylicacid cycle andbecause of the Keq of aconitaselead to a spilling over of citrate.Unfortunately, none of the explanations which are based on an inhibition of NAD-or NADP-specific isocitrate dehydrogenases have ever been supported by evidence from in vivoexperiments. The glycerol theory (Legisa and Mattey, 1986; Gradisnik-Grapulin and Legisa,1996), has recently been reassessed by studying the effect of increased mycelial glycerolconcentrations on the oxidation of 1,514C-citrate by mycelia and isolated mitochondria ofA. niger (Arisan-Atac and Kubicek, 1996). The appearance of 14C-labelled CO2whichbecause of the labelling position applied can only be released during the metabolic conversionof citrate to a-ketoglutaratewas virtually unaffected by the glycerol concentration, therebyclearly disproving an effect of glycerol on the activity of isocitrate dehydrogenases andconsequently this theory. Also, in contrast to the enzyme from crude cell-free extracts (Legisaand Mattey, 1986), the purified NADP-specific isocitrate dehydrogenase was not inhibitedby citrate (Arisan-Atac and Kubicek, 1996).

It is surprising that the question of whether the isocitrate dehydrogenase step of the TCAcycle is active during citric acid fermentation or not has never been viewed from a theoreticalpoint of view: using the cellular concentration of free and protein-bound glutamic acid asan indicator of metabolic flux from glucose to a-ketoglutarate, there is no indication for asignificant change in this flux unless at late stages of fermentation where the fungal growth(and also the need for glutamic acid) has stopped, and this flux is only 17 per cent lowerthan that occurring in a culture accumulating 78 per cent less citric acid, and hence may notbe of high relevance to the mechanism of citric acid accumulation (O.Zehentgruber andC.P.Kubicek, unpublished data).

With regard to the mechanisms which trigger the initial accumulation of citrate from themitochondria, a fact completely overlooked so far is the activity of the tricarboxylatetransporter. This carrier competes directly with aconitase for citrate, and if its affinity forcitrate were much higher than that of aconitase, would pump citrate out of the mitochondriawithout any necessity for inhibition of one of the TCA cycle enzymes. As the tricarboxylatecarrier of mammalian tissues and yeast occurs by countertransport with malate (Evans et


al., 1983), such a situation is conceivable when its counter-ion malate accumulates in thecytosol. Malate accumulation has in fact been shown to precede citrate accumulation (Roehrand Kubicek, 1981). However, the mitochondrial citrate carrier of A. niger has not yet beeninvestigated, and this hypothesis clearly needs thorough investigation before it can be usedto explain citrate accumulation. It is also not known to what extent changes in the fluxthrough the NAD-dependent-, NADP-dependent isocitrate dehydrogenases, a-ketoglutaratedehydrogenase and succinate dehydrogenase, contribute to a rise in the intramitochondrialcitrate concentration. As these enzymes are known to be regulated by the mitochondrialNADH/NAD and NADPH/NADP ratios, as well as by AMP, cis-aconitate and oxaloacetate(Chan et al., 1965; Meixner-Monori et al., 1985, 1986), fluctuations in the level ofmitochondrial TCA metabolites are likely.

2.4.2 Respiratory activity and the role of NAD regeneration

Formation of citric acid is dependent on strong aeration, and dissolved oxygen tensionshigher than those required for vegetative growth of A. niger stimulate citric acid fermentation(Clark and Lentz, 1961; Kubicek et al., 1980; Dawson et al., 1988a). On the other hand,sudden interruptions in the air supply cause an irreversible impairment of citric acidproduction without any harmful effect on mycelial growth (Kubicek et al., 1980; Dawson etal., 1988b). The biochemical basis of this observation appears to be related to the presenceof an alternative respiratory pathway, which is obviously required for re-oxidation of theglycolytically produced NADH, by a continuously maintained, high oxygen tension (Kubiceket al., 1980; Zehentgruber et al., 1980; Kirimura et al., 1987, 1996), whose activity is impairedby short interruptions in the air supply (Kubicek et al., 1980). Weiss and colleagues (Wallrathet al., 1991; Schmidt et al., 1992; Prmper et al., 1993) studied the role of the standard andalternative respiratory pathways in citric acid accumulation in detail. They detected that theassembly of the proton pumping NADH:ubiquinone oxidoreductase is impaired during citricacid accumulation (Schmidt et al., 1992), which could be the reason for the importance ofthe activity of the alternative pathway. Interestingly, disruption of the gene encoding theNADH-binding subunit of complex I in a low producing strain of A. niger increased itscatabolic overflow, yet this strain excreted much less citrate than its parent (Prmper et al.,1993). These findings stress the fact that citric acid accumulation is not a mono-causalprocess, and citrate accumulation in high amounts depends on a delicate balance of severalfactors, whose interrelationship is not yet fully understood.

The requirement of a high oxygen supply is also related to another effect of Mn2+ ions onA. niger, i.e. on the morphology of the fungus: whereas A. niger grows in long and smoothfilaments when supplied with optimal concentrations of Mn2+ ions, Mn2+ deficient grownmycelia are strongly vacuolated, highly branched, contain strongly enthickened cell wallsand exhibit a bulbous appearance (Kisser et al., 1980; Papagianni et al., 1994). This type ofmorphology has been shown to provide a much better rheology (Olsvik et al., 1993) andenables a higher oxygen transfer (Fujita et al., 1994; Iwahori et al., 1995); it may thus berequired for optimal citric acid yields.

NAD regeneration may also be related to the effect of pH on citric acid fermentation:the almost quantitative conversion of glucose to citric acid, as occurs during the idiophaseof fermentation, yields 1 ATP and 3 NADH. While part of the NADH pool can be reoxidizedby the alternative, salicylhydroxamic acid (SHAM) sensitive respiratory pathway describedabove, this yield of ATP probably still exceeds that of the cells maintenance demands.Roehr et al. (1992) speculated that the ATP will be consumed by the plasma membrane


bound ATPase during maintenance of the pH gradient between the cytosol and theextracellular medium. The involvement of this enzyme in the maintainance of the pHgradient in citric acid producing A. niger has been shown by Mattey et al. (1988). Hencethe requirement of a low pH for citric acid accumulation may be, at least in part, relatedto a high turnover of the ATP formed, which otherwise would lead to a metabolic imbalanceand so stop acidogenesis. However, this explanation still requires experimental verification.Most recently, single-point mutagenesis of a plant ATPase and its expression in yeastresulted in increased H+-pumping and increased growth rates at low pH (Morsomme etal., 1996).

2.5 Export of citric acid from A. niger

Torres et al. (1994a) proposed that the two citric acid transport steps, i.e. that from themitochondria to the cytosol, and that from mycelia into the culture filtrate, are among themost important regulatory points for the obtention of high yields.

The mechanism of transport of mitochondrial citrate into the cytosol is still completelyunknown, except for the hypothesis that it occurs by countertransport with theglycolytically overproduced malate (see above). ATP-citrate lyase, an enzyme which inother cells uses the cytosolic citrate for lipid biosynthesis, appears to be unable to managethis high efflux but its precise regulation under citric acid producing conditions is notunderstood (Pfitzner et al., 1987; Jernejc et al., 1991). The latter authors have also purifieda cytosolic and a mitochondrial carnitine acetyltransferase from A. niger, which exhibitedsimilar kinetic and physicochemical properties (Jernejc and Legisa, 1996). As the activityof this enzyme was in considerable excess of that of ATP-citrate lyase they concluded thattransfer as a carnitine ester may be the major physiological source of acetyl-CoA for lipidbiosynthesis. If this is indeed the case it would explain why the cytosolic citrate pool israther stable. Because of their findings of a cytosolic isoenzyme of carnitineacetyltransferase, Jernejc and Legisa (1996) also speculated that this enzyme transfersacetyl-CoA to the mitochondria and thus for citrate biosynthesis. This is an intriguingspeculation, but requires the identification of a cytosolic pathway from pyruvate to acetyl-CoA which is not yet known.

Mattey and co-workers (Mattey, 1992; Kontopidis et al., 1995) explained the export ofcitrate through the plasma membrane in terms of the large pH gradient between the cytosoland the extracellular medium, and postulated that citrate efflux from the cells may occur bydiffusion of the 2(-) citrate anion, driven by a gradient. If this assumption is correct, the lowpH would be responsible for the citrate gradient necessary for transport and consequentlyless citrate would be secreted at higher pH values. However, recent studies in our laboratoryclearly showed that citrate export requires ATP, and its Vmax is not strongly affected by theexternal pH (Netik et al., 1997); this renders the diffusion hypothesis rather unlikely. Netiket al. (1997) also reported that citrate export is strongly increased in mycelia grown undermanganese deficiency, which is consistent with previous observations that the intracellularconcentration of citrate in manganese sufficient and deficient grown mycelia is not greatlydifferent (Kubicek and Roehr, 1985; Legisa and Kidric, 1989; Prmper et al., 1993), despitethe five- to seven-fold higher extracellular levels under the latter conditions. The reason forthe requirement of manganese deficiency for citrate export is not clearly understood, butmay be related to an absolute requirement of citrate uptake for manganese ions, probablybecause of a requirement for chelated citrate as a substrate for the permease (Netik et al.,1997).


The reason for the reciprocal effect of Mn2+ ions on export and import of citric acid mayalso be related to yet another effect of manganese deficiency, i.e. inhibition of triglycerideand phospholipid synthesis as well as a shift in the ratio of saturated to unsaturated fattyacids of whole mycelial lipids (Orthofer et al., 1979; Jernejc et al., 1989) and of isolatedplasma membranes (Meixner et al., 1985). The different behaviour of the citrate export andimport system of A. niger may also be seen in the light of earlier studies on the antagonismof several membrane affecting compounds on the detrimental action of manganese ions,e.g. lower alcohols (Moyer, 1953), lipids (Millis et al., 1963; Gold and Kieber, 1967), ortertiary amines (Batti, 1969). Also the technically important ability of Cu2+ ions to antagonizethe deleterious effect of Mn2+ may be related to citrate excretion, as Cu2+ strongly inhibitedthe uptake of citric acid from the medium (Netik et al., 1997). However, the effect of Cu2+(Schweiger, 1959) may also reside in its inhibition of the uptake of Mn2+ by A. niger (Hockertzet al., 1987), which occurs by a specific, high affinity transport system (Seehaus et al.,1990). The properties of the uptake and the export system are otherwise similar (?pH drivenproton symport) and it may be speculated that they are catalyzed by the same enzymesystem.

2.6 References

AHMED, S A, SMITH, J E and ANDERSON, J G, 1972. Mitochondrial activity during citric acidproduction by Aspergillus niger, Transactions of the British Mycological Society, 59, 5161.

ARISAN-ATAC, I and KUBICEK, C P, 1996. Glycerol is not an inhibitor of mitochondrial citrateoxidation in Aspergillus niger, Microbiology UK, 142, 29372942.

ARISAN-ATAC, I, WOLSCHEK, M and KUBICEK, C P, 1996. Trehalose-6-phosphate synthase Aaffects citrate accumulation by Aspergillus niger under conditions of high glycolytic flux,FEMS Microbiology Letters, 140, 7783.

ARTS, E, KUBICEK, C and ROEHR, M, 1987. Regulation of phosphofructokinase from Aspergillusniger: effect of fructose-2,6-bisphosphate on the action of citrate, ammonium ions and AMP,Journal of General Microbiology, 133, 11951199.

BAKER, H V, 1986. Glycolytic gene expression in Saccharomyces cerevisiae: nucleotide sequenceof GCR1, null mutations, and evidence for expression, Molecular and Cellular Biology, 6,37743784.

BAKER, H V, 1991. GCR1 of Saccharomyces cerevisiae encodes a DNA binding protein whosebinding is abolished by mutations in the CTTCC sequence motif, Proceedings of the NationalAcademy of Sciences of the USA, 88, 94439447.

BATTI, M A, 1969. US Patent 3,438.863.BERCOVITZ, A, PELEG, Y, BATTAT, E, ROKEM, J S and GOLDBERG, I, 1990. Localisation of

pyruvate carboxylase in organic acid producing Aspergillus strains, Applied and EnvironmentalMicrobiology, 56, 15941597.

BERRY, D R, CHMIEL, A and AL-OBAIDY, Z, 1977. In: Genetics and Physiology of Aspergillus.Eds J E SMITH and J A PATEMAN (Academic Press, London), pp. 405423.

BLOOM, S J and JOHNSON, M J, 1962. The pyruvate carboxylase of Aspergillus niger, Journal ofBiological Chemistry, 237, 27182720.

BODDY, L M, BERGES, T, BARREAU, C, VAINSTAIN, M H, JOBSON, M J, BALLANCE, D Jand PEBERDY, J F, 1993. Purification and characterization of an Aspergillus niger invertaseand its DNA sequence, Current Genetics, 24, 6066.

BOWES, I and MATTEY, M, 1979. A study of mitochondrial NADP+-specific isocitratedehydrogenase from selected strains of Aspergillus niger, FEMS Microbiology Letters, 7, 323325.


BRINDLE, P K, HOLLAND, J P, WILLET, C E, INNIS, M A and HOLLAND M J, 1990. Multiplefactors bind the upstream activation sites of the yeast endolase genes ENO1 and ENO2: ABF1protein, like repressor activator protein RAP1, binds cis-acting sequences which modulaterepression or activation of transcription, Molecular and Cellular Biology, 10, 48724895.

CHAMBERS, A, STANWAY, C, TSANG, J S H, HENRY, Y, KINGSMAN, A J and KINGSMAN,S M, 1990, ARS binding factor 1 binds adjacent to RAP1 at the UASs of the yeastglycolyticgenes PGK and PYK1, Nucleic Acids Research, 18, 53935399.

CHAN, M F, STACHOV, C S and SANWAL, B D, 1965. The allosteric nature of NAD-specificisocitric dehydrogenase of Aspergilli, Canadian Journal of Biochemistry, 43, 111118.

CHASMAN, D I, LUE, N F, BUCHMAN, A R, LAPOINTE, J W, LORCH, Y and KORNBERG, RD, 1990. A yeast protein that influences the chromatin structure of UASG and functions as apowerful auxiliary gene activator, Genes and Development, 4, 503514.

CHOE, J, and YOO, Y J, 1991. Effect of ammonium ion concentration and application to fed-batchculture for overproduction of citric acid, Journal of Fermentation and Bioengineering, 72,106109.

CLARK, D S and LENTZ, C P, 1961. Submerged citric acid fermentation on beet molasses: effectof pressure and recirculation of oxygen, Canadian Journal of Microbiology, 7, 447452.

CLARK, D S, ITO, K and HORITSU, H, 1966. Effect of manganese and other heavy metals onsubmerged citric acid fermentation of molasses, Biotechnology and Bioengineering, 8, 465471.

CLELAND, W W and JOHNSON, M J, 1954. Tracer experiments on the mechanism of citric acidformation by Aspergillus niger, Journal of Biological Chemistry, 208, 679692.

CLEMENTS, J M and ROBERTS, C F, 1986. Transcription and processing signals in the 3-phosphoglycerate kinase (PGK) gene from Aspergillus nidulans, Gene, 44, 97105.

DAWSON, M W, MADDOX, I S, BOAG, I F, and BROOKS, J D, 1988a. Application of fed-batchculture to citric acid production by Aspergillus niger: the effects of dilution rate and dissolvedoxygen tension, Biotechnology and Bioengineering, 32, 220226.

DaWSON, M W, MADDOX, I S, and BROOKS, J D, 1988b. Effect of interruptions to the airsupply on citric acid production by Aspergillus niger, Enzyme and Microbial Technology, 8,3740.

DE GRAAFF, L and VISSER, J, 1988. Structure of the Aspergillus nidulans pyruvate kinase gene,Current Genetics, 14, 553560.

DE GRAAFF, L, VAN DEN BROECK, H and VISSER, J, 1988. Isolation and characterisation ofthe pyruvate kinase gene of Aspergillus nidulans, Current Genetics, 13, 315321.

DE GRAAFF, L, VAN DEN BROECK, H and VISSER, J, 1992. Isolation and characterisation ofthe Aspergillus niger pyruvate kinase gene, Current Genetics, 22, 2127.

DRONAWAT, S N, SVIHLA, C K and HANLEY, T R, 1995. The effects of agitation and aerationon the production of gluconic acid by Aspergillus niger, Applied Biochemistry and Biotechnology,51/52, 347354.

EVANS, C T, SCRAGG, A H and RATLEDGE, C, 1983. A comparative study of citrate efflux frommitochondria of oleaginous and non-oleaginous yeasts, European Journal of Biochemistry,130, 195204.

FALKNER, F G, MOCIKAT, R and ZACHAU, H G, 1986. Sequences closely related to animmunoglobulin gene promoter/enhancer element occur also upstream of other eukaryotic andof prokaryotic genes, Nucleic Acids Research, 14, 88198827.

FEIR, H A and SUZUKI, I, 1969. Pyruvate carboxylase of Aspergillus niger: kinetic study of abiotin-containing enzyme, Canadian Journal of Biochemistry, 47, 697710.

FIEDUREK, J, SZCZODRAK, J and ILCZUK, Z, 1988. Citric acid synthesis by Aspergillus nigermutants resistant to 2-deoxy-D-glucose: decreased synthesis with biomass increase, ActaMicrobiologica Polonica, 36, 303307.

FOSTER, J W, 1949. Chemical Activities of Fungi (Academic Press, London).FUJITA, M, IWAHORI, K, TATSUTA, S and YAMAKAWA, K, 1994. Analysis of pellet formation

of Aspergillus niger based on shear stress, Journal of Fermentation and Bioengineering, 78,368373.


GOLD, W and KIEBER, R J, 1967. Process of making citric acid by fermentation, US Patent337,2094.

GRADISNIK-GRAPULIN, M and LEGISA, M, 1996. Comparison of specific metaboliccharacteristics playing a role in citric acid excretion between some strains of the genusAspergillus, Journal of Biotechnology, 45, 265270.

GUPTA, S, and SHARMA, C B, 1995. Citric acid fermentation by the mutant strain of the Aspergillusniger resistant to manganese ions inhibition, Biotechnology Letters, 17, 269274.

HABISON, A, KUBICEK, C P and ROEHR, M, 1979. Phosphofructokinase as a regulatory enzymein citric acid accumulating Aspergillus niger, FEMS Microbiology Letters, 5, 3942.

HABISON, A, KUBICEK, C P and ROEHR, M, 1983. Partial purification and regulatory propertiesof phosphofructokinase from Aspergillus niger, Biochemical Journal, 209, 669676.

HARMSEN, H, KUBICEK-PRANZ, E M, VISSER, J, ROEHR, M and KUBICEK, C P, 1992.Regulation of 6-phosphofructo-2-kinase from the citric acid producing fungus Aspergillus niger,Applied Microbiology and Biotechnology, 37, 784787.

HAYASHI, S and NAKAMURA, S, 1981. Multiple forms of glucose oxidase with differentcarbohydrate compositions, Biochimica et Biophysica Acta, 657, 4051.

HOCKERTZ, S, PLNZIG, J and AULING, G, 1987. Impairment of DNA formation is an earlyevent in Aspergillus niger under manganese starvation, Applied Microbiology and Biotechnology,25, 590593.

HOCKERTZ, S, SCHMID, J and AULING, G, 1987. A specific transport system for manganese inthe filamentous fungus Aspergillus niger, Journal of General Microbiology, 133, 35133519.

HONECKER, S, BISPING, B, YANG, Z and REHM, H-J, 1989. Influence of sucrose concentrationand phosphate limitation on citric acid production by immobilized cells of Aspergillus niger,Applied Microbiology and Biotechnology, 31, 1724.

HOSSAIN, M, BROOKS, J D and MADDOX, I S, 1984. The effect of sugar source on citric acidproduction by Aspergillus niger, Applied Microbiology and Biotechnology, 19, 383391.

HUIE, M A, SCOTT, E W, DRAZINIC, C M, LOPEZ, M C, HORNSTRA, I K, YANG, T P andBAKER, H V, 1992. Characterization of the DNA-binding activity of GCR1: in vivo evidencefor two GCR1-binding sites in the upstream activating sequences of TPI of Saccharomycescerevisiae, Molecular and Cellular Biology, 12, 26902700.

HUSSAIN, M, RAHMAN, R, CHOUDHURY, N and HUSSAIN, I, 1978. Studies on mitochondrialrespiration of some high citric acid-yielding mutants of Aspergillus niger, Journal ofFermentation Technology, 56, 253256.

IWAHORI, K, TATSUTA, S, FUJITA, M and YAMAKAWA, M, 1995. Substrate permeability inpellets formed by Aspergillus niger, Journal of Fermentation and Bioengineering, 79, 387390.

JAKLITSCH, W M, KUBICEK, C P and SCRUTTON, M C, 1991. Intracellular organisation ofcitrate production in Aspergillus niger, Canadian Journal of Microbiology, 37, 823827.

JERNEJC, K and LEGISA, M, 1996. Purification and properties of carnitine acetyltransferase fromcitric acid producing Aspergillus niger, Applied Biochemistry and Biotechnology, 60, 151158.

JERNEJC, K, VENDRAMIN, M and CIMERMAN, A, 1989. Lipid composition of Aspergillusniger in citric acid accumulating and nonaccumulating conditions, Enzyme and MicrobialTechnology, 11, 452456.

JERNEJC, K, PERDIH, A and CIMERMAN, A, 1991. ATP:citrate lyase and carnitineacetyltransferase activity in a citric-acid-producing Aspergillus niger strain, AppliedMicrobiology and Biotechnology, 36, 9295.

KIRIMURA, K, HIROWATARI, Y and USAMI, S, 1987. Alterations of respiratory systems inAspergillus niger under the conditions of citric acid fermentation, Agricultural BiologicalChemistry, 51, 12991303.

KIRIMURA, K, SARANGBIN, S, RUGSASEEL, S and USAMI, S, 1992. Citric acid productionby 2-deoxyglucose-resistant mutant strains of Aspergillus niger, Applied Microbiology andBiotechnology, 36, 573577.


KIRIMURA, K, MATSUI, T, SUGANO, S and USAMI, S, 1996. Enhancement and repression ofcyanide-insensitive respiration in Aspergillus niger, FEMS Microbiology Letters, 141, 251254.

KISER, R-C and NIEHAUS, W G JR, 1981. Purification and kinetic characterization of mannitol-1-phosphate dehydrogenase from Aspergillus niger, Archives of Biochemistry and Biophysics,211, 613621.

KISSER, M, KUBICEK, C P and ROEHR, M, 1980. Influence of manganese on morphology andcell-wall composition of Aspergillus niger during citric acid fermentation, Archives ofMicrobiology, 128, 2633.

KONTOPIDIS, G, MATTEY, M and KRISTIANSEN, B, 1995. Citrate transport during the citricacidfermentation by Aspergillus niger, Biotechnology Letters, 17, 11011106.

KUBICEK, C P, 1988. The role of the citric acid cycle in fungal organic acid fermentations.Biochemical Society Symposia, 54, 113126.

KUBICEK, C P and ROEHR, M, 1980. Regulation of citrate synthase from the citric acid producingfungus Aspergillus niger, Biochimica et Biophysica Acta, 615, 449457.

KUBICEK, C P and ROEHR, M, 1985. Aconitase and citric acid accumulation in Aspergillus niger,Applied and Environmental Microbiology, 50, 13361338.

KUBICEK, C P and ROEHR, M, 1986. Citric acid fermentation. CRC Critical Reviews inBiotechnology, 3, 331373.

KUBICEK, C P, HAMPEL, W A and ROEHR, M, 1979a. Manganese deficiency leads to elevatedamino acid pools in citric acid accumulating Aspergillus niger, Archives of Microbiology, 123,7379.

KUBICEK, C P, ZEHENTGRUBER, O and ROEHR, M, 1979b, An indirect method for studyingfine control of citric acid accumulation by Aspergillus niger, Biotechnology Letters, 1, 4752.

KUBICEK, C P, ZEHENTGRUBER, O, EL-KALAK, H and ROEHR, M, 1980. Regulation ofcitric acid production by oxygen: effects of dissolved oxygen tension on adenylate levels andrespiration in Aspergillus niger, European Journal of Applied Microbiology and Biotechnology,9, 101116.

KUBICEK, C P, SCHREFERL-KUNAR, G, WHRER, W and ROEHR, M, 1988. Evidence for acytoplasmic pathway of oxalate biosynthesis in Aspergillus niger, Applied and EnvironmentalMicrobiology, 54, 633637.

KUBICEK-PRANZ, E M, MOZELT, M, ROEHR, M and KUBICEK, C P, 1990. Changes in theconcentration of fructose2,6-bisphosphate in Aspergillus niger during stimulation ofacidogenesis by elevated sucrose concentration, Biochimica et Biophysica Acta, 1033, 250255.

LA NAUZE, J M, 1966. Aconitase and isocitric acid dehydrogenases in Aspergillus niger in relationto citric acid accumulation, Journal of General Microbiology, 44, 7381.

LEGISA, M and BENCINA, M, 1994, Evidence for the activation of 6-phosphofructo-1-kinase bycAMP-dependent protein kinase in Aspergillus niger, FEMS Microbiology Letters, 118, 327334.

LEGISA, M and GRADISNIK-GRAPULIN, M, 1995. Sudden substrate dilution induces a higherrate of citric acid production by Aspergillus niger, Applied and Environmental Microbiology,61, 27322737.

LEGISA, M and KIDRIC, J, 1989. Initiation of citric acid accumulation in the early stages ofAspergillus niger growth, Applied Microbiology and Biotechnology, 31, 453457.

LEGISA, M and MATTEY, M, 1986, Glycerol as an initiator of citric acid accumulation in Aspergillusniger, Enzyme and Microbial Technology, 8, 607609.

LEGISA, M and MATTEY, M, 1988. Citrate regulation of the change in carbohydrate degradationduring the initial phase of citric acid production by Aspergillus niger, Enzyme and MicrobialTechnology, 10, 3336.

MA, H, KUBICEK, C P and ROEHR, M. 1981, Malate dehydrogenase isoenzymes in Aspergillusniger, FEMS Microbiology Letters, 12, 147151.

MA, H, KUBICEK, C P and ROEHR, M, 1985. Metabolic effects of manganese deficiency inAspergillus niger: evidence for increased protein degradation, Archives of Microbiology, 141,266268.


MACHIDA, M, GONZALEZ, T V J, BOON, L K, GOMI, K and JIGAMI, Y, 1996. Molecularcloning of a genomic DNA for enolase from Aspergillus oryzae, Bioscience, Biotechnologyand Biochemistry, 60, 161163.

MARTIN, S M and WILSON, P W, 1951. Uptake of 14CO2 by Aspergillus niger in the formation ofcitric acid, Archives of Biochemistry, 27, 150157.

MATTEY, M, 1977. Citrate regulation of citric acid production by Aspergillus niger. FEMSMicrobiology Letters, 2, 7174.

MATTEY, M, 1992. The production of organic acids, CRC Critical Reviews in Biotechnology, 12,87132.

MATTEY, M, LEGISA, M and LOWE, S, 1988. Effect of lectins and inhibitors on membranetransport in Aspergillus niger, Biochemical Society Transactions, 16, 969970.

MCKNIGHT, G L, OHARA, P J and PARKER, M L, 1986. Nucleotide sequence of thetriosephosphate isomerase gene from Aspergillus nidulans: implications for a differential lossof introns, Cell, 46, 143147.

MCNEIL, J B, DYKSHOORN, P, HUY, J N and SMALL, S, 1990. The DNA-binding proteinRAP1 is required for efficient transcriptional activation of the yeast PYK glycolytic gene,Current Genetics, 18, 405412.

MEIXNER, O, MISCHAK, H, KUBICEK, C P and ROEHR, M, 1985. Effects of manganesedeficiency on plasma membrane lipid composition and glucose uptake in Aspergillus niger,FEMS Microbiology Letters, 26, 271274.

MEIXNER-MONORI, B, KUBICEK, C P and ROEHR, M, 1984. Pyruvate kinase from Aspergillusniger: a regulatory enzyme in glycolysis? Canadian Journal of Microbiology, 30, 1622.

MEIXNER-MONORI, B, KUBICEK, C P, HABISON, A, KUBICEK-PRANZ, E M and ROEHR,M, 1985. Presence and regulation of the a-ketoglutarate dehydrogenase multienzyme complexin the filamentous fungus Aspergillus niger, Journal of Bacteriology, 161, 265271.

MEIXNER-MONORI, B, KUBICEK, C P, HARRER, W, SCHREFERL, G and ROEHR, M, 1986.NADP-specific isocitrate dehydrogenase from the citric acid accumulating fungus Aspergillusniger, Biochemical Journal, 236, 549557.

MILLIS, N F, TRUMPY, B H and PALMER, B M, 1963. The effect of lipids on citric acid productionby an Aspergillus niger mutant, Journal of General Microbiology, 30, 365379.

MISCHAK, H, KUBICEK, C P and ROEHR, M, 1985. Formation and location of glucose oxidasein

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